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Phosphoric acid receptor protein gene glandular related viral vector and construction and application

A virus vector, phosphoprotein technology, applied in application, gene therapy, genetic engineering and other directions, can solve the problems of adenovirus contamination application, obstacles and other problems, achieve the effect of wide application, avoid immune response, and improve cardiac function

Inactive Publication Date: 2005-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional preparation of recombinant AAV is to use adenovirus-assisted transfection method, but the problem of adenovirus contamination is an obstacle to its application

Method used

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  • Phosphoric acid receptor protein gene glandular related viral vector and construction and application
  • Phosphoric acid receptor protein gene glandular related viral vector and construction and application
  • Phosphoric acid receptor protein gene glandular related viral vector and construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of gene drug rAAV-asPLB

[0044] According to the PLB gene sequence and the plasmid pAAV-MCS structure, design the PLB gene primers with BamHI and EcoRI restriction sites (lowercase letters in the sequence below), and the upstream primer sequence of the PCR reaction is: 5'-cgggatccCGCAGCTGAGCTGAGCTCCCAGACTTCA-3', The downstream primer sequence is: 5'-cggaattcTTTAAATTTCATTTATTCCCAA-3'. The PLB cDNA fragment (695bp) was amplified by PCR reaction, connected to the plasmid pAAV-MCS, and the plasmid pAAV-asPLB plasmid was constructed, and the insertion direction and sequence correctness were identified by enzyme digestion and sequencing. Take 10 μg each of the plasmids pAAV-asPLB, pAAV-RC and pHelper, and transfect 293 cells by calcium phosphate co-precipitation method to construct the recombinant PLB gene antisense RNA adeno-associated virus vector (rAAV-asPLB); at the same time, set up a positive control, namely pAAV-LacZ 293 cells were co-transfec...

Embodiment 2

[0045] Example 2: Application of recombinant virus rAAV-asPLB in cultured neonatal mouse cardiomyocytes

[0046] Take 1-3 days old rat ventricular muscle, cut into 1mm 3 Left and right small pieces, repeated digestion with 0.125% trypsin, filtered through 200-mesh steel mesh, centrifuged at 1000rpm for 5min to collect cells, 5% CO 2 Incubate at 37°C for 1 hour in a cell culture incubator to remove adherent cells. Resize cells to 1 x 10 6 / ml inoculated in 6-well plates. According to the virus-to-cell ratio (MOI) of 100, the recombinant virus was added to infect the cultured rat cardiomyocytes. The results showed that the recombinant virus rAAV-asPLB inhibited the mRNA transcription and protein translation of the PLB gene in cultured neonatal mouse cardiomyocytes; decreased the cytoplasmic Ca 2+ ion concentration, to reduce calcium overload, see Figure 4 , Figure 5 . Figure 4 RT-PCR detection of PLB mRNA transcription in cardiomyocytes infected with recombinant virus,...

Embodiment 3

[0047] Example 3: Recombinant virus rAAV-asPLB is used to study the changes in the amount and activity of calcitonin in diabetic rats

[0048]After the rats were weighed, intraperitoneal injection of streptozotocin (STZ) solution according to the dose of 65mg / kg, the successful production of diabetic rats in a clean environment and feeding for 6 weeks, intraperitoneal injection of chloral hydrate (400mg / kg) for general anesthesia , take the supine position, open the trachea, connect the animal ventilator, open the chest cavity, and expose the heart. Draw 100 μL of recombinant virus (about 1×10 9 IU) Inject 50 μL of the recombinant virus into the apex of the heart and the wall of the left ventricle. Close the chest and remove the ventilator. After waking up, the rats were fed with ordinary feed in a clean environment. The results showed that the recombinant virus rAAV-asPLB inhibited the mRNA transcription and protein translation of PLB gene in cardiomyocytes of diabetic rat...

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Abstract

The present invention provides the construction and application of phosphoric acid accepting protein gene glandular associated viral vector. The construction process includes 1) amplifying PLB gene segment; 2) constructing and identifying antisense PLB recombinant plasmid vector pAAV-asPLB; 3) preparing glandular associated viral vector rAAV-asPLB; and 4) collecting, purifying, identifying and titrating glandular associated viral vector rAAV-asPLB. During the construction of the vector, AAV virus gene is eliminated to avoid immulogical reaction and other side effects the virus causes, and the related plasmid, etc. are pharmaceutically acceptable material. The present invention is safe, wide in application range and high in infecting efficiency, has long term stable intracorporeal expression of treating gene, and may be used in preparing gene medicine for treating heart incompetence and diabetes.

Description

technical field [0001] The present invention relates to biotechnology, mainly relates to phosphoacceptance protein (phosphoceptin, phospholamban, phospholamban) gene antisense RNA recombinant adeno-associated virus vector (rAAV-asPLB, rAAV-asPLN, rAAV-asPL) and construction, and in Application in the preparation of drugs for diseases such as cardiovascular disease, diabetic cardiomyopathy, and abnormal calcium regulation. Background technique [0002] With the development of the Human Genome Project and the gradual deepening of molecular biology research, the molecular mechanisms of many diseases have been elucidated and the causative genes have been continuously discovered. Gene therapy has developed from the treatment of single-gene genetic diseases to the treatment of polygenic common and frequently-occurring diseases. , especially in the treatment of cardiovascular and cerebrovascular diseases and various tumors, showing good application prospects. Three key issues in g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P3/10A61P9/00C12N15/10C12N15/12C12N15/861
Inventor 胡申江李江
Owner ZHEJIANG UNIV
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